In living cells, the uptake and extrusion of hydrophilic molecules are generally governed by specialized membrane proteins known as transporters or carriers. Unlike pores or channels, they undergo enzyme-like binding and conformational changes to promote energetically downhill transport of their specific molecules. Elucidation of the catalytic properties of carriers by a combination of experimental measurements of carrier fluxes and analysis of those data by kinetic models is a fundamental endeavor in this field. This chapter provides a systematic guideline towards the formulation and analysis of kinetic models of carrier-mediated transport across biomembranes. Detailed analyses are provided for the transport of Ca2+ ions into the mitochondrial matrix via the Ca2+uniporter located in the inner mitochondrial membrane. A brief introduction is also given for the kinetic treatment of cotransporters and antiporters. The analyses presented in this chapter can be easily extended to study the kinetics of carrier-mediated ion and metabolite transport in general.